We describe the adaptation of a hybrid quadrupole linear ion trap-orbitrap mass spectrometer to accommodate electron-transfer ion/ion reactions (ETD) for peptide and protein characterization. The method utilizes pulsed, dual electrospray ion sources and requires minimal instrument modification. Switching between cation and reagent anion injection schemes is automated and accomplished within a few hundred milliseconds. Ion/ion reactions are conducted within the linear ion trap, after which the c- and z-type product ions are passed to the orbitrap for high-resolution m/z analysis. With this arrangement, mass accuracies are typically measured to within 2 ppm at a resolving power of approximately 60 000. Using large peptides and intact proteins, we demonstrate such capabilities will accelerate our ability to interrogate high-mass species. To illustrate compatibility with automated data-dependent analysis and subsequent data processing, we couple the technique with an online chromatographic separation of a yeast whole-cell lysate followed by peptide identification using ProSight PC. Fairly long pulsing times and relatively low ET efficiency, as compared to conventional ETD instrumentation, are the main drawbacks of this approach. Still, our results suggest that the implementation of ETD on sensitive, high-resolution, and high-mass accuracy hybrid instrumentation, such as the orbitrap, will substantially propel the emergent fields of middle- and top-down proteomics.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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